Solutions of Quaternary and Tertiary Ammonium Cation Detergents to Denature Proteins, and Kits for Making Them

ABSTRACT

Ammonium cation detergents comprising a quaternary or tertiary ammonium cation can be used as detergents to denature proteins and are particularly useful in denaturing glycoproteins or glycopeptides prior to enzymatic deglycosylation. Ammonium cation detergents with sulfate or sulfonate anions are particularly useful.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/376,342, filed Aug. 17, 2016. The contents ofthis application are incorporated herein by reference for all purposes.

STATEMENT OF FEDERAL FUNDING

Not applicable.

BACKGROUND OF THE INVENTION

This invention relates to the field of analysis of glycosylation ofglycoproteins.

Many of the proteins produced by eukaryotic cells are modified aftertranslation by the addition of covalently-linked, linear or branchedchains of carbohydrates. These protein-carbohydrate conjugates arereferred to as glycoproteins; the point at which the carbohydrate isattached is referred to as a glycosylation site. Attachedpolysaccharides or oligosaccharides are referred to as glycans. A widerange of glycans are found on the different glycosylation sites ofparticular glycoproteins. The particular pattern of glycans on aparticular glycoprotein is determined by the specific cell line thatproduced the protein and the conditions under which the cells weregrown.

Since the glycans conjugated to a protein can affect characteristicscritical to its function, including pharmacokinetics, stability,bioactivity, or immunogenicity, it is important in many uses todetermine which glycans are present. Thus, the ability to remove some orall of the glycans from a protein and to analyze the glycans or theprotein, or both, to determine their composition or compositions isuseful for determining whether a protein will have a desired effect. Forexample, the Food and Drug Administration requires characterization ofcarbohydrates attached to biologics (such as therapeutic glycoproteinsand vaccines) to show composition of matter and consistency ofmanufacture, resulting in a need for extensive characterization of theproduct. Analysis of the profile of the released carbohydrates is alsoimportant for quality control in the production of recombinant proteins,in which a change in carbohydrate profile may indicate stress in thesystem, signaling conditions that may require a commercial-scalefermenter of expensive protein to be discarded.

The techniques used to analyze glycans are complex, cumbersome and oftentime-consuming. The glycans must be released or removed from theprotein, a process known as “deglycosylation”, before analysis can beperformed. Further, the samples to be analyzed typically include celllysates, cell culture supernatants, and clinical plasma or serumsamples, which may contain a multitude of glycoproteins in addition tothe one of interest. Thus, companies wishing to obtain analysis of thecarbohydrates attached to a particular glycoprotein, such as an antibodyintended for therapeutic use in humans, often have first to perform anumber of steps to isolate the target glycoprotein from others in theraw sample.

Only a subset of proteins can be deglycosylated under native, ornon-denaturing, conditions, in which the protein is simply mixed with anenzyme that will release from the protein the glycans conjugated to theprotein. These methods have the advantage of mild conditions and simpleclean up, but often result in incomplete release of glycans. For mostproteins being deglycosylated by enzymatic digestion, however, thesecondary and tertiary structures of the proteins do not permit accessof the enzyme to the carbohydrates unless the protein is first denaturedto alter those structures. Traditional protocols for denaturing involvethe use of detergents and reducing agents, and an overnight incubation.For example, these protocols typically add to the glycoprotein areducing agent such as beta-mercaptoethanol, an anionic detergent, suchas sodium dodecyl (lauryl) sulfate, a non-ionic detergent, such asoctylphenolpoly(ethyleneglycolether), and a deglycosylating enzyme, andincubating the resulting mixture for 16 hours at 37° C. Once the proteinis deglycosylated, the glycans are removed and, usually, are labeled.These protocols are effective and largely independent of the protein(that is, they can be used on most proteins), but are harsh, typicallyuse detergents which must be removed before some analytical processescan be conducted, and can have a number of clean-up steps.

In commercial cell culture settings, the length of sample processing,typically at least a day, when these standard deglycosylation methodsare used reduces the possibility of using carbohydrate analysis as amarker for rapid, in-process analysis and as a tool for control ofprocess variables, such as stress during the cell culture process. Theuse of detergents adds steps and time since any detergent remaining withthe released glycans can interfere with the results of mass spectrometryor other techniques used to analyze the carbohydrate profile

Release of the glycans from the glycoprotein is typically achieved byone of two methods, chemical release or enzymatic digestion. Most of theavailable methods for chemical release result in the destruction of theprotein backbone of the glycoprotein, and are therefore unsuitable whenanalysis of the protein component of the glycoprotein is desired.Enzymatic digestion usually occurs under milder conditions and leavesthe protein component intact. It is therefore preferable in manyanalytical situations. Enzymatic digestion is particularly useful forremoving N-glycans (glycans linked to the protein through amide groupsof asparagine residues), which can be released from glycoproteins byenzymatic cleavage using the exemplar enzyme PNGase F(Peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase, EC 3.5.1.52) orendoglycosidases such as endo-alpha-N-acetyl-galactosaminidase,Endoglycosidase F1, Endoglycosidase F2, Endoglycosidase F3, orEndoglycosidase H. The glycans are then typically treated to label theirfree-reducing terminus with a fluorescent dye, excess label in removed,and the labeled glycans analyzed by methods such as high performanceliquid chromatography (HPLC), capillary electrophoresis (CE), orcarbohydrate gel electrophoresis. The protein component can be analyzedby any of various techniques, including mass spectrometry (MS). Bothenzymatic digestion and chemical deglycosylation procedures encompassmultiple steps, extended incubation times, and clean-up steps prior toanalysis of the released glycans.

More recently, various protocols have been developed which reduce thetime needed for deglycosylation compared to traditional protocols. In2010, Agilent Technologies announced the introduction of a so-called“mAb-Glyco Chip” for deglycosylation and analysis of monoclonalantibodies, in which a deglycosylation enzyme is immobilized in a thincapillary in which the monoclonal antibodies of interest are flowed.Agilent's product materials stated that the system allowsdeglycosylation of the monoclonal antibodies in four minutes. In anotherexample, U.S. Patent Publication No. 20130171658 describes methods ofreleasing glycans from a target glycoprotein in a biological sample inwhich the biological sample is added to a solid support comprising anaffinity ligand immobilized in a packed bed, on a monolith, or on amembrane, binding the target glycoprotein, washing away any unboundglycoprotein, and then contacting the bound target with adeglycosylation enzyme to release glycans from the glycoprotein. Thisprotocol permits rapid isolation and deglycosylation of a targetglycoprotein even if other glycoproteins in the sample are not known.These protocols are based, in part, on improving the kinetics of theenzymatic deglycosylation by placing the glycoprotein into closeproximity to an immobilized enzyme, as compared to traditional protocolsin which both the enzyme and the glycoprotein are in solution and reactby solution-phase kinetics.

Reagent and device manufacturers have also tried to find reagents whichreduce the number of steps or time to complete an analytical workflow,U.S. Pat. No. 7,229,539 discloses degradable surfactants which do notneed to be removed following the deglycosylation reaction. According tothe patent, once deglycosylation has taken place, the surfactant isdegraded by adding an acid to the medium and the resulting products arecompatible with MS.

There remains a need for additional methods of deglycosylation that canbe performed rapidly, that reduce handling and clean up steps, and thatare suitable for use with multiple analytic methods, particularly withmass spectrometry. Surprisingly, the present invention meets these andother needs.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, the invention provides methods of denaturing aglycoprotein or glycopeptide of interest in vitro, comprising incubatingthe glycoprotein or glycopeptide in vitro with a solution comprising aneffective amount of an ammonium cation sulfate or sulfonate detergentcomprising (a) an aliphatic chain of 8-24 carbons, (b) a sulfate orsulfonate anion and (c) a tertiary or quaternary ammonium cation,wherein said aliphatic chain is covalently attached to said anion, for atime T to denature said glycoprotein or glycopeptide, thereby denaturingsaid glycoprotein or glycopeptide. In some embodiments, the anion is asulfate anion. In some embodiments, the anion is a sulfonate anion. Insome embodiments, the chain of 8-24 carbons is saturated. In someembodiments, the aliphatic chain is of 8-15 carbons. In someembodiments, the aliphatic chain is of 8-13 carbons. In someembodiments, the aliphatic chain is 12 carbons, In some embodiments, thealiphatic chain is saturated. In some embodiments, the aliphatic chainis in a ring configuration. In some embodiments, the sulfate orsulfonate anion is covalently attached to said aliphatic chain by beingattached to a benzyl which is attached to the aliphatic chain. In someembodiments, the cation is a quaternary ammonium cation. In someembodiments, the quaternary ammonium cation is selected from the groupconsisting of:

In some embodiments, the quaternary ammonium cation is atetramethylammonium cation. In some embodiments, the quaternary ammoniumcation is a tetrabutyl, a tetraethyl, or a tetrapropyl ammonium cation.In some embodiments, the ammonium cation detergent is tetramethyldodecyl sulfate. In some embodiments, the cation is a tertiary ammoniumcation. In some embodiments, the tertiary ammonium cation is atrimethyl, tributyl, triethyl or tripropyl ammonium cation. In someembodiments, the glycoprotein or glycopeptide of interest is in amixture of glycoproteins. In some embodiments, the glycoprotein orglycopeptide of interest is in a cell lysate, blood serum, blood plasma,is a fusion protein, or is a cell membrane protein. In some embodiments,the method further comprises the steps of heating a solution comprisingthe glycoprotein or glycopeptide and the ammonium cation sulfatedetergent or the ammonium cation sulfonate detergent to a temperatureranging from about 80° to about 120° C., maintaining the mixture withinthe temperature range for time T, and then cooling said solution. Insome embodiments, the temperature range is from about 90° to about 100°C. In some embodiments, the solution is cooled to a temperature of about35-60° C. following time T. In some embodiments, the solution is cooledto a temperature of about 50° C. In some embodiments, time T is betweenabout 1 to about 10 minutes. In some embodiments, time T is about 3minutes. In some embodiments, the method further comprises releasingglycans from the denatured glycoprotein or glycopeptide by incubatingthe denatured glycoprotein or glycopeptide with a deglycosylation enzymefor a time sufficient to release said glycans, thereby forming asolution comprising the glycans, the glycoprotein or glycopeptide fromwhich said glycans have been released, and the ammonium cation sulfateor sulfonate detergent. In some embodiments, either the glycoprotein orglycopeptide or the deglycosylation enzyme is immobilized on a solidsupport. In some embodiments, the denatured glycoprotein or glycopeptideis immobilized on a solid support prior to being contacted with thedeglycosylation enzyme. in some embodiments, the deglycosylation enzymeis an amidase. In some embodiments, the amidase is PNG F. In someembodiments, the released glycans are labeled following release from theglycoprotein or glycopeptide. In some embodiments, the released glycansare released as β-glycosylamines. In some embodiments, the label isfluorescent. In some embodiments, the labeled released glycans areanalyzed. In some embodiments, the analysis is selected from the groupconsisting of high-performance liquid chromatography, hydrophilicinteraction chromatography, nuclear magnetic resonance, fluorescenceanalysis, Western blotting, gel electrophoresis, capillaryelectrophoresis, microfluidic separation, and mass spectrometry or acombination of any two or more of these. In some embodiments, theanalyzing is by detecting a fluorescent signal from said labeledreleased glycans. In some embodiments, the denatured glycoprotein orglycoprotein (now deglycosylated) from which said glycans have beenreleased is analyzed. In some embodiments, the analyzing of theglycoprotein or glycoprotein from which glycans have been released is byhigh-performance liquid chromatography, hydrophilic interactionchromatography, nuclear magnetic resonance, Western blotting, gelelectrophoresis, fluorescence analysis, capillary electrophoresis,microfluidic separation, mass spectrometry, or a combination of two ormore of any of these. In some embodiments, the analysis is by massspectrometry. In some embodiments, the solution further comprises areductant, an alkylant, an additional organic solvent denaturant, achaotrope, or a combination of any of these. In some embodiments, theammonium cation sulfate detergent or ammonium cation sulfonate detergentis removed prior to the analyzing. In some embodiments, the removing ofthe ammonium cation sulfate detergent or the ammonium cation sulfonatedetergent comprises flowing the solution comprising the releasedglycans, the glycoprotein or glycoprotein from which the glycans havebeen released, and the detergent into a solid phase extraction device toremove the detergent from the solution.

In a further group of embodiments, the invention provides methods ofdenaturing a glycoprotein or glycopeptide of interest in vitro,comprising incubating the glycoprotein or glycopeptide in vitro with asolution comprising effective amounts of (a) a compound of the formula:R-Y-.M+, in which: R is a saturated or unsaturated straight chainaliphatic group with 8-13 carbon atoms; Y− is a sulfate or sulfonateanion; M+ is a cation of an element in periodic table group 1; and,thedot indicates that the cation is ionically, not covalently, associatedwith the Y− anion, and either (b) an ammonium cation detergentcomprising: (i) an aliphatic chain of 8-24 carbons, (ii) a carboxylateanion covalently attached to the aliphatic chain and, (iii) a tertiaryor quaternary ammonium cation ionically, not covalently, associated withthe carboxylate anion, or (b′) a molar excess of quaternary ammoniumcations, or of tertiary ammonium cations, or of both, In someembodiments, R is an aliphatic group having 12 carbons. In someembodiments, Y− is a sulfate anion. In some embodiments, the tertiary orquaternary ammonium cation of said ammonium cation detergent is aquaternary ammonium cation, In some embodiments, the ammonium cationdetergent is tetramethylammonium acetate or tetramethyiammonium laurate.In some embodiments, the molar excess of said quaternary ammoniumcations or tertiary ammonium cations is of quaternary ammonium cations.In some embodiments, the quaternary ammonium cation is selected from thegroup consisting of:

In some embodiments, the quaternary ammonium cation is tetramethylammonium. In some embodiments, the molar excess of quaternary ammoniumcations is of tetramethyl ammonium cations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 is a table showing the comparative results of studiesusing sodium dodecyl sulfate and tetramethyl ammonium lauryl sulfate asdetergents in a deglycosylation protocol on a series of glycoproteinsand mixtures of glycoproteins. To permit ready comparison of how welleach detergent served in the deglycosylation protocol, the amount ofglycans released from each glycoprotein or mixture of glycoproteins inthe assay in which tetramethyl ammonium dodecyl sulfate was used as thedetergent was normalized to 100%, and the amount released then comparedto the amount of glycan released when SDS was used as the detergent inthe same deglycosylation protocol on the same glycoprotein. hIgG:mixture of human IgG antibodies. hIgA: mixture of human IgA antibodies.hIgM: mixture of human IgM antibodies. “Nist mAb”: An antibody providedby the National Institute of Standards and Technology (“NIST”), asreference material 8671.

FIG. 2. FIG. 2 presents the results of the comparative studies reportedin FIG. 1 in the form of a graph. The error bars represent the resultsof three replicates. The “tetramethylammonium dodecyl sulfate” referredto on FIG. 2 is the same compound referred to as “tetramethyl ammoniumlauryl sulfate” in FIG. 1.

DETAILED DESCRIPTION

Which carbohydrates, or glycans, are attached to a glycoprotein isimportant to understanding the pharmacokinetics, immunogenicity, andpotential therapeutic effectiveness of the glycoprotein. Accordingly,removing the glycans and analyzing them to determine which glycans areattached to a given glycoprotein has become an important aspect ofquality control for glycoproteins, such as antibodies and otherbiologics, intended for therapeutic use. Further, analyzing theaglycosylated glycoprotein after its glycans have been removed has alsobecome important for confirming the composition of the aglycosylatedprotein.

Unfortunately, some glycoproteins, especially some antibody andantibody-derived therapeutics, are resistant to enzymaticdeglycosylation by standard protocols, especially those involvingrelease of N-glycans by the exemplar enzyme PNGase F. Since there is noway to determine in advance whether any particular glycoprotein ofinterest is easy or hard to deglycosylate by enzymatic release of theglycans, it is generally determined empirically by subjecting theglycoprotein to an enzymatic deglycosylation protocol intended to removeN-glycans, O-glycans, or both, known to be present on the glycoproteinand analyzing the molecular weight of the protein following theprocedure to determine whether the molecular weight indicates that theN-glycans, O-glycans, or both, present on the glycoprotein have beenremoved or that some remain attached. Glycoproteins are typicallydenatured prior to enzymatic deglycosylation so that the enzyme hasaccess to glycosylation sites which otherwise might be hidden or blockedby the protein's secondary or tertiary structure. Often, a detergent isused to solubilize the glycoprotein as part of the denaturation step.The detergent may also participate in denaturation of the glycoprotein,and may be referred to as a denaturant in addition to being referred toas a detergent. It would be convenient to have additional detergentsthat can work across a range of glycoproteins from those relativelyeasy-to-deglycosylate to those relatively hard-to-deglycosylate in anattempt to reduce the number of glycoproteins for which protocols haveto be adopted.

Experiments were performed in attempts to find detergents that would beeffective in enzymatic deglycosylation protocols over a range ofglycoproteins. As antibodies and antibody-derived glycoproteins areincreasingly important as therapeutic agents, some of our studiesfocused on examining the deglycosylation of a selection of some of thecurrently-available antibody and anti-derived agents.

In initial studies underlying the present invention, we found that fattyacid salts with alkali metal cations, such as sodium laurate, were gooddetergents for deglycosylation of many glycoproteins. We further foundthat substituting an exemplar quaternary ammonium cation, tetramethylammonium, in place of the alkali metal, sodium, in sodium laurate,resulted in deglycosylation about as good as that when using sodiumlaurate for many glycoproteins, but in markedly better deglycosylationof some glycoproteins. It was found, for example, that in the presenceof the exemplar quaternary ammonium cation fatty acid salt, adeglycosylation protocol resulted in the release of 21% more glycan fromIgM than did the same protocol using the fatty acid salt, but with analkali metal cation rather than the quaternary ammonium cation. Thus, wefound that quaternary ammonium fatty acid salts are superior to fattyacid salt detergents for denaturing glycoproteins for enzymaticdeglycosylation protocols, and particularly for deglycosylationprotocols for antibodies and antibody-derived glycoproteins. Referencesherein to “quaternary ammonium carboxyl salts” or “quaternary ammoniumfatty acid salts” or “quaternary ammonium fatty acid detergents” referto compounds with a quaternary ammonium cation, and an aliphatic chainattached to a carboxylate anion.

Surprisingly, when we tested an exemplar quaternary ammoniumcation-sulfate detergent, tetramethylammonium dodecyl sulfate, with asulfate anion replacing the laurate of the corresponding quaternaryammonium cation fatty acid detergent, we got markedly better resultsthan were obtained with the exemplar quaternary ammonium fatty acid saltin deglycosylating glycoproteins.

The detergents were tested on a recombinant fusion proteinZiv-aflibercept, a cancer therapeutic sold under the name ZALTRAP®. Topermit ready comparison, the glycans released by the exemplar quaternaryammonium-sulfate detergent, tetramethylammonium dodecyl sulfate, weretreated as representing 100% release of glycans from Ziv-aflibercept,and that amount was compared to the amount of glycans released fromZiv-aflibercept using the same protocol and the corresponding exemplarquaternary ammonium fatty acid salt detergent, tetramethylammoniumdodecyl laurate. Use of the quaternary ammonium fatty acid saltdetergent resulted in the release of 80-90% of the amount of glycansreleased from ZALTRAP® by the quaternary ammonium-sulfate detergent.Tests with other hard-to-deglycosylate glycoproteins confirmed that theexemplar quaternary ammonium—fatty acid detergent tested was not assuccessful at deglycosylating such glycoproteins as the exemplarquaternary ammonium-sulfate detergent. Accordingly, we concluded thatquaternary ammonium-sulfate detergents are surprisingly betterdetergents for denaturing glycoproteins for enzymatic deglycosylationprotocols than are either quaternary ammonium—fatty acid detergents orfatty acid detergents.

Sodium dodecyl sulfate (“SDS,” also known as sodium lauryl sulfate) is adetergent widely used to denature and solubilize proteins, particularlybefore subjecting them to electrophoresis, and is commonly used todenature glycoproteins in deglycosylation protocols. As a denaturant fordeglycosylation applications, SDS is often used in combination with anon-ionic surfactant, typically NP-40, after the denaturing step butprior to adding the enzyme to prevent denaturation of the enzyme. Aseries of studies was conducted to determine how the exemplar quaternaryammonium-sulfate detergent, tetramethyl ammonium dodecyl sulfate,compared to SDS.

In the studies reported herein, nine exemplar glycoproteins or mixturesof glycoproteins were subjected to enzymatic deglycosylation followingdenaturing using either an exemplar quaternary ammonium-sulfatedetergent, or SDS. To allow ready comparison, the amount of glycansreleased from each glycoprotein or mixture by digestion followingdenaturing with tetramethyl ammonium lauryl sulfate was considered to bea 100% release, and that amount was then compared to the amount releasedfrom the same glycoprotein or glycoprotein mixture following denaturionwith SDS. Surprisingly, for four out of nine of the glycoproteins orglycoprotein mixtures, denaturing with SDS resulted in the release ofsignificantly fewer glycans than did the use of the exemplar quarternaryammonium sulfate detergent, and for a mixture of human IgGs, denaturingwith SDS resulted in the release of almost 25% less glycan than diddenaturing with the quarternary ammonium sulfate detergent. For amixture of hIgM antibodies, denaturing with SDS resulted in the releaseof 18% less glycan than did denaturing with the quarternary ammoniumsulfate detergent. For the therapeutic biologic drug ORENCIA®,denaturing with SDS resulted in the release of almost 15% less glycanthan did denaturing with the quarternary ammonium sulfate detergent. Forthree of the nine glycoproteins or mixtures, using SDS to denature theglycoprotein resulted in about the same release of glycan compared withthe amount released following denaturation of the same glycoprotein withthe quarternary ammonium sulfate detergent. Only in the case of oneglycoprotein did denaturing with SDS result in the release of moreglycan than did the use of quarternary ammonium sulfate detergent, andin that case the difference was less than 10%. The results show that theexemplar quarternary ammonium sulfate detergent provided generallybetter or equal results to using SDS in the deglycosylation protocol,and in many cases, provided surprisingly better results compared SDS,both for some hard-to-denature glycoproteins and for mixtures ofglycoproteins.

As noted above, enzymatic deglycosylation protocols in which SDS is usedas a detergent typically recite the addition of a non-ionic detergentprior to introducing the enzyme to avoid denaturing it. Studies with anexemplar quaternary ammonium-sulfate detergent revealed that nonon-ionic detergent was needed. Indeed, the studies showed that thepresence of a non-ionic detergent is preferably not used. Thus,embodiments of the inventive methods allow eliminating a reagent used intypical protocols.

Given the results with an exemplar quarternary ammonium sulfatedetergent, it is believed that other detergents which provide aquaternary ammonium cation, an aliphatic chain, and a sulfate anion willalso be surprisingly useful detergents for denaturing glycoproteinscompared to detergents with an aliphatic chain and a sulfate anion, buta counterion different from a quaternary ammonium cation or a tertiaryammonium cation, as further discussed below. It is further believedthat, given the structural and functional similarity of a sulfonateanion to a sulfate anion, that detergents with a quarternary ammoniumcation, and an aliphatic chain covalently attached to a sulfonate anionwill also be surprisingly useful detergents for the same purposes. Giventhe difference in charge between the sulfate and the sulfonate anions,we believe that, between detergents differing only between a sulfateversus a sulfonate anion, the one with the sulfate anion will be thebetter detergent. Further, given the structural and functionalsimilarity of a tertiary ammonium cation to a quaternary ammoniumcation, we expect tertiary ammonium cations to form effective detergentswith sulfate or sulfonate anions. In some embodiments, the detergentshave a quarternary ammonium cation, an aliphatic chain, and a sulfateanion.. In some embodiments, the detergents have a quarternary ammoniumcation, an aliphatic chain, and a sulfonate anion. In some embodiments,the detergents have a tertiary ammonium cation, an aliphatic chain, anda sulfate anion. In some embodiments, the detergents have a tertiaryammonium cation, an aliphatic chain, and a sulfonate anion.

As noted in the Background, once the glycoproteins or glycopeptides aredenatured, they are frequently subjected to enzymatic digestion torelease N-linked carbohydrates as glycosylamines, which are thentypically labeled with an amine-reactive dye. Primary and secondaryammonium salts are less preferred for use in the inventive methodsbecause they could compete with glycans released as glycosylaminesfollowing enzymatic digestion and reduce the availability of theglycosylamines for labeling. Tertiary and quaternary ammonium sulfateand sulfonate detergents are expected to be useful for denaturing theglycoprotein but not to react with glycosylamines released from theglycoproteins or glycopeptides of interest by enzymatic digestion. Toavoid constant repetition of “glycoprotein or glycopeptide,” referencesto deglycosylation or analysis of a “glycoprotein” herein encompassdeglycosylation or analysis of a “glycopeptide” unless otherwiserequired by context.

Ammonium Cation Sulfate and Sulfonate Detergents

As reported above, an exemplar quaternary ammonium sulfate detergent,was surprisingly more effective in denaturing glycoproteins than a likequaternary ammonium detergent based on a fatty acid, and then thecommonly used detergent SOS. These results indicate that effectivedetergents can be made following the formula of the following formula:

R-Y⁻-⋅C+, in which:   Formula 1

-   R is a saturated or unsaturated aliphatic group with 8-22 carbon    atoms;-   Y⁻ is sulfate or sulfonate;-   C⁺ is a quaternary ammonium cation or a tertiary ammonium cation;    and,-   the dot indicates that the cation is ionically, not covalently    associated, with the Y⁻.

In preferred embodiments, the aliphatic group is saturated. In someembodiments, Y⁻ is sulfate. In some embodiments. Y⁻ is sulfonate. Insome embodiments in which Y⁻ is a sulfate or sulfonate, the aliphaticgroup is 8-20, 9-19, 9-18, 9-17, 9-16, 10-16, 10-15, 10-15, 11-14,11-13, or 12 carbon atoms in length, with each succeeding range ornumber stated being more preferred than the one preceding it. Analiphatic group of twelve carbons is particularly preferred. Thealiphatic group is preferably straight. In some embodiments, it can bein a ring configuration. The aliphatic group is covalently attached tothe Y⁻. As used herein, the phrase “ammonium sulfate detergent” or“ammonium sulfonate detergent” refers to a detergent of Formula 1. Asused herein, the phrases “quaternary ammonium sulfate detergent” and“quaternary ammonium sulfonate detergent” refer to a detergent ofFormula 1 in which C⁺ is a quaternary ammonium cation. As used herein,the phrases “tertiary ammonium sulfate detergent” and “tertiary ammoniumsultanate detergent” refer to a detergent of Formula 1 in which C⁺ is atertiary ammonium cation.

Quaternary ammonium cations have the advantage of maintaining theirpositive charge independent of the pH of their environment, whereas thecharge of primary, secondary and tertiary ammonium cations can varyaccording to the surrounding pH. It is anticipated that mostdeglycosylation procedures and other denaturing procedures will beconducted at pHs at which tertiary ammonium cations will maintain theirpositive charge and can be useful as cations of compounds of Formula 1.For example, the ammonium cation can be a tertiary ammonium cation, suchas trimethyl ammonium, triethyl ammonium, tripropyl ammonium or tributylammonium. Primary and secondary ammonium cations are expected to be lessuseful as cations for detergents, as the nitrogen may be accessible toreact with amine-reactive dyes or other reagents.

In some embodiments, the cation is a quaternary ammonium cation. As usedherein, “quaternary ammonium cation” refers to a moiety having theformula N⁺¹R₁R₂R₃R₄, wherein each R can be the same or different and arechosen from aryl or alkyl, can be saturated or unsaturated, can beunsubstituted or substituted, may contain atoms other than carbon orhydrogen in the chain or ring or attached to the chain or ring,including carbon-bonding substituents such as sulfur, oxygen, nitrogen,boron, or a halogen, and functional groups containing any of these, andcan be another quaternary group. In some preferred embodiments, thequaternary ammonium cation is selected from the group consisting of:

Cations with a smaller number of atoms are generally preferred overcations with a larger number of atoms.

In some embodiments, the cation can instead be a tertiary ammoniumcation. As used herein, a “tertiary ammonium cation” refers to a moietyhaving the formula HN⁺¹R₁R₂R₃, wherein R₁, R₂, and R₃, can be the sameor different and are chosen from aryl or alkyl, can be saturated orunsaturated. can be unsubstituted or substituted, may contain atomsother than carbon or hydrogen in the chain or ring or attached to thechain or ring, including carbon-bonding substituents such as sulfur,oxygen, nitrogen, boron, or a halogen, and functional groups containingthese, and can be another tertiary group.

Fatty acid salts of ammonium cations which are not commerciallyavailable can be synthesized by reacting the fatty acid of choice withthe hydroxide conjugate base. For example, tetramethylammonium lauratecan be synthesized by reacting lauric acid with tetramethyl ammoniumhydroxide and we made tetraethyl ammonium laurate, tetrapropyl ammoniumlaurate, and tetrabutyl ammonium laurate by the same process. Thehydroxide conjugate bases of some, if not all, of the quaternaryammonium cations listed are commercially available. Any that are not canbe synthesized by methods well known in the art, such as reacting a saltof the quaternary ammonium cation with a strong base. For example,tetramethyl ammonium hydroxide is typically made by mixingtetramethylammonium chloride and potassium hydroxide in dry methanol.

Ammonium cation sulfate and sulfonate detergents can be made by anyconvenient method known in the art, such as by acid basedneutralization, as discussed above, or by ion exchange. A procedure forpreparing an exemplar quaternary ammonium sulfate cation detergent byion exchange is set forth in the Examples.

Combinations of ammonium salts and periodic table group 1 detergents

It is believed that sulfate or sulfonate detergents having a cation ofan element of periodic table group 1 can be used in combination withquaternary ammonium cations and tertiary ammonium cations to formunexpectedly powerful detergents for denaturing glycoproteins fordeglycosylation. The quaternary ammonium cations or tertiary ammoniumcations can be contributed by, for example, a quaternary ammonium fattyacid salt.

These detergents comprising elements in periodic table group 1 have thefollowing formula:

R-Y-⋅M+, in which:   Formula 2

-   R is a saturated or unsaturated straight chain aliphatic group with    8-13 carbon atoms;-   Y− is a sulfate or sulfonate anion;-   M+ is a cation of an element of periodic table group 1; and,-   the dot indicates that the cation is ionically, rather than    covalently, associated with the Y− anion.

It is believed compounds of this formula will be good detergents to bemixed with one or more quaternary or tertiary ammonium cation salts, orquaternary or tertiary ammonium cation cations, for denaturingglycoproteins or glycopeptides in deglycosylation protocols. Inpreferred embodiments, the aliphatic group is saturated. In preferredembodiments, the aliphatic group is 10-12 carbons in length. In someembodiments, Y− is a sulfate anion. In some embodiments, Y− is asulfonate anion. For convenience of reference, compounds of Formula 2may also sometimes be referred to herein as “M+ detergents.”

Given the structural similarity of tertiary ammonium salts to quaternaryammonium salts, it is also expected that tertiary ammonium salts can beused with a M+ detergent of Formula 2 to achieve similar results. Anexcess of quaternary or tertiary ammonium cations can be provided by anyconvenient means known in the art. Primary and secondary ammonium saltsare less preferred because they could compete with glycans released asglycosylamines during a subsequent enzymatic digestion and thus wouldrequire an additional cleanup step in a deglycosylation protocol toremove them after the denaturing step but before the enzymatic digestionstep,

In some embodiments, the solution contains quaternary ammonium cations.In some embodiments, the solution comprises a molar excess of quaternaryammonium cations compared to the compound of Formula 2. In someembodiments, the solution contains tertiary ammonium cations. In someembodiments, the solution contains a molar excess of tertiary ammoniumcations to the compound of Formula 2.

Use of the mixtures and detergents described above allows methods ofreleasing glycans from glycoproteins that reduce the time needed forworkflows to release glycans from glycoproteins and then to analyze thereleased glycans, the protein from which the glycans have been released,or both.

Mixtures of M⁺ detergents and Ammonium Cation Salts for Use In PreparingGlycoproteins for Deglycosylation Protocols

It is believed a mixture of a compound of Formula 2 and a quaternaryammonium salt in a deglycosylation protocol allows rapid and effectiveenzymatic deglycosylation of a range of antibody and antibody-derivedglycoproteins. The effective deglycosylation is expected to be due atleast in part to the detergent effect of the mixture in allowing accessof PNGase F to sites at which N-glycans are attached to the protein. Itis further believed that this is due in part to the interaction of thequaternary ammonium cation with the sulfated aliphatic chain contributedby the compound of Formula 2 to the reaction mixture at the pH of thesolution.

Salts of other quaternary ammonium cations can contribute quaternaryammonium cations to the mixture and result in effective deglycosylationof a range of glycoproteins. Such salts are suitable so long as they arecapable of dissociating under the pH and temperature conditions to beutilized in the deglycosylation procedure. Whether any particular saltcan be used in the inventive methods can be readily determined by simplyrunning a deglycosylation protocol in parallel on two aliquots of thesame glycoprotein, preferably an antibody or an antibody-derivedglycoprotein, with the protocol differing only in the detergent mixused, and comparing the amounts and types of glycans released from eachaliquot. Test salts that are suitable for use in the inventive methodswill result in the release of at least all of the same glycans as arereleased by the reference salt, in amounts equal to, greater than, ornot more than 5% less than the amounts released by the reference mix.

Due to their structural and functional similarity to quaternary ammoniumcations, it is believed that salts of tertiary ammonium cations can alsocontribute ammonium cations to the mixture and result in effectivedeglycosylation of a range of glycoproteins. Secondary and primaryammonium cations are believed likely to work for effectivedeglycosylation, but are less preferred because they could interferewith later labeling of released glycans unless additional cleanup stepsare introduced into the protocol.

Mixtures of Detergents of Formula 1 and of Formula 2

It is believed that combining a detergent of Formula 2 with an ammoniumcation sulfate or sulfonate detergent of Formula 1 will be useful inunfolding glycoproteins while keeping them in solution, rendering siteson the glycoprotein accessible to the deglycosylation enzyme that mightnot be made accessible by either detergent used on its own. Accordingly,in some embodiments, mixtures of detergents of Formula 1 and of Formula2 are used in the enzymatic deglycosylation of glycoproteins, andparticularly of antibodies and antibody-derivatives.

Ammonium Fatty Acid Salts

As noted above, initial studies using a fatty acid salt bearing anexemplar quaternary ammonium cation, proved to be good detergent fordenaturing and deglycosylating glycoproteins. The exemplar ammoniumfatty acid salt tested, tetramethyl ammonium laurate, caused the releaseof as much glycan as did an exemplar fatty acid salt, sodium laurate,when used in deglycosylation protocols on a number of glycoproteins, butresulted in releasing significantly more glycan from somehard-to-deglycosylate proteins. A comparison was made of the relativeability of tetramethyl ammonium laurate and an exemplar alkali metalfatty acid salt, sodium laurate, to serve as a detergent in denaturingglycans in an exemplar deglycosylation protocol. The detergents weretested on 18 different glycoproteins, including a number of FDA-approvedtherapeutic antibodies, and the resulting released glycans wereanalyzed. Previous work with similar data sets indicated that greaterthan a 5% difference in glycan release is significant.

The quaternary ammonium salt tetramethyl ammonium laurate and the alkalimetal salt sodium laurate were also tested as detergents on complexmixtures of glycoproteins. First, the detergents were tested on celllysates of Chinese hamster ovary (“CHO”) cells. Cell lysates contain,among other glycoproteins, membrane proteins. As noted by Waas et al.,Anal. Chem., 2014, 86(3):1551-1559, membrane proteins are hard tosubject to enzymatic digestion due to their hydrophobic properties.Second, the detergents were tested on mammalian blood serum. In bothcases, the exemplar alkali metal fatty acid salt and glycoproteinmixture precipitated after the denaturation, interfering with therelease of glycans, while the quaternary ammonium fatty acid salt andglycoprotein mixture remained in solution and allowed enzymatic releaseof glycans present in the sample.

Based on the results with two fusion proteins and two complexglycoprotein mixtures, ammonium cation fatty acid detergents were betterin deglycosylation protocols for fusion proteins, particularly thosecontaining Fc portions of antibodies, and for complex glycoproteinmixtures (including those containing membrane proteins) than are alkalimetal or alkali earth metal fatty acid detergents, while working as wellas alkali metal or alkali earth metal fatty acid detergents indeglycosylation protocols for glycoproteins such as antibodies

The glycosylamines were released by enzymatic digestion and were thenlabeled with an amine-reactive dye. Embodiments in which the denaturantis present during the deglycosylation step is advantageous because ithelps prevent the glycoprotein from refolding and perhaps rendering somesites once again inaccessible to the enzyme before deglycosylation canoccur.

Ammonium Cation Detergents

As noted, our initial studies involved detergents having a quaternaryammonium cation and a fatty acid chain terminating in a carboxylate (an“ammonium carboxylate detergent”). We then discovered that an exemplardetergent with a quaternary ammonium cation and a sulfate anion wassurprisingly better than the like detergent with a carboxylate anion.Accordingly, in some embodiments, the inventive compositions, methods,and kits employ a tertiary or quaternary ammonium cation and a sulfateor sulfonate anion. In other embodiments, the inventive compositions,methods, and kits employ a compound of Formula 2 and a tertiary orquaternary ammonium cation detergent, such as a quaternary ammoniumsulfate detergent or a quaternary ammonium carboxylate detergent.

The initial studies conducted with an exemplar quaternary ammoniumcarboxylate detergent showed it was useful in denaturing complexmixtures of glycoproteins and other proteins found in cell lysates andmammalian blood serum. Given the surprisingly better results indeglycosylating glycoproteins and mixtures of glycoproteins that weobtained using an exemplar quaternary ammonium sulfate detergentcompared to the exemplar quaternary ammonium carboxylate detergent, itis believed that tertiary ammonium sulfate detergents and quaternaryammonium sulfate detergents, and tertiary ammonium sultanate andquaternary ammonium sulfonate detergents, will likewise provesurprisingly more useful in denaturing complex mixtures of glycoproteinsand other proteins found in cell lysates and mammalian blood serum thanare tertiary or quaternary ammonium carboxylate detergents.

It is expected that tertiary ammonium sulfate and sulfonate detergentsand quaternary ammonium sulfate and sulfonate detergents, andparticularly tertiary or quaternary ammonium sulfate detergents, willremain soluble with a somewhat longer aliphatic chain than fattyacid-based detergents, and thus in some embodiments may be 8-22 carbonslong. In some embodiments, the aliphatic chain may be 8-21 carbons long,in some embodiments may be 8-20 carbons long, in some embodiments may be8-19 carbons long, in some embodiments may be 8-18 carbons long, in someembodiments may be 8-17 carbons long and in some embodiments may be 8-16carbons long, while in still others, the chain may be 8-15 carbons long,in some embodiments may be 8-14 carbons long, and in some otherembodiments may be 8-13 carbons long. In some preferred embodiments, thealiphatic chain is saturated. In some embodiments, the aliphatic chainis unsaturated.

Lauric acid, which has 12 carbons, is one preferred for making ammoniumcation sulfate and sulfonate detergents, with fatty acids with 11carbons being next preferred. In some embodiments, the fatty acid usedto form the salt has 12 carbons and is saturated. In some embodiments,the fatty acid used to form the salt has 11 carbons and is saturated. Itis noted that “lauric acid” and the other trivial names for fatty acidsrefer to acids bearing the carboxylic acid end. Ammonium cation sulfateand sulfonate detergents derived from such fatty acids are oftenreferred to by the cation, the length of the aliphatic chain, and theanion, such as “triethanol dodecanoic sulfate,” “trimethanol decanoicsulfonate”, or tributyl tetradecanoic sulfate.” This practice is notuniversally followed as, for example, “ammonium lauryl sulfate” is ananionic surfactant commonly found as an ingredient of shampoos and bodywashes, while the IUPAC name can state the chain length first, then theammonium cation, then the anion, as in “octadecyl trimethyl ammoniumsulfate” or “hexadecyl-trimethylammonium sulfonate.” The particularnomenclature chosen is not important.

Branched fatty acid chains do not seem to be useful. In someembodiments, however, the fatty acid can have 8-13 carbons and be in aring configuration.

Use of Formula 1 and Formula 2 Detergents as Reagents to Denature otherProteins

As reported in the Examples, an exemplar quaternary ammonium sulfatedetergent proved surprisingly useful in denaturing glycoproteins andglycoprotein mixtures as part of a deglycosylation protocol. Theseresults indicate that tertiary and quaternary ammonium sulfate andsulfonate detergents, will also be useful reagents for otherproteolytic, analytic, and diagnostic protocols, such as in denaturingproteins prior to conducting a Western blot, and in denaturing a fusionprotein or glycoproteins or proteins in a complex mixture, such as acell lysate or blood serum or plasma. Tertiary and quaternary ammoniumsulfate and sulfonate detergents are expected to be particularly usefulfor denaturing cell membrane proteins.

Concentrations

Typically, mixtures in which a compound of Formula 2 is mixed with aquaternary or tertiary ammonium cation salt, such as an ammonium cationfatty acid salt, will be 0.50% to 3.0%, 0.75-2.50%, 0.75.-2.25%,1.0-2.0%, or 1.0-1.50% Formula 2 detergent, with each successive rangebeing more preferred to the one before it. Good results can be obtainedusing a concentration of 125%, which concentration is the mostpreferred. The quaternary or tertiary ammonium cation salt is preferablypresent at 10 mM-150 mM, 10-125 mM, 20-100 mM, 25-100 mM, 30-100 mM.30-90 mM. 30-80 mM. 30-75 mM, 40-70 mM, 40-65 mM, 40-60 mM, or 45-55 mM.Good results were obtained using 50 mM of exemplar quaternary ammoniumdetergents. The mixtures were in an aqueous base. The pH can be between6 and 9.5 and may be a pH of 7-9, or of 8-9, Some of the underlyingstudies were performed in solutions without significant bufferingcapacity, at a pH of 8.5, which is a preferred pH for some embodiments.

In embodiments in which a quaternary or tertiary ammonium sulfate orsulfonate (Formula 1) detergent is used, the detergent will typically beused in a concentration of 0.01 to 2.5%, more preferably 0.05 to about2.0%, still more preferably 0.1 to about 1.5%, and in still morepreferred embodiments, 0.01 to about 1%, where the term “about” meansplus or minus 0.05% and the concentration is measured as volume/volume.Persons of skill are aware that glycoproteins differ in how hard theyare to denature and that how hard any particular glycoprotein is todenature is usually determined empirically. If a particular glycoproteinproves to be hard to denature, the concentration of the detergent can beat the higher end of the stated amounts. As glycoproteins that are hardto denature are typically denatured at a higher temperature, ahard-to-denature glycoprotein will usually also be subjected to a highertemperature.

Use of Multiple Detergents to Provide More Complete Solubilization

Glycoproteins are usually denatured in a buffer which already containsone or more salts. For example, phosphate buffered saline is a commonbuffer which, as its name states, is a saline solution. Somecombinations of ammonium cation sulfate or sulfonate detergent incombination with the salt already in the buffer, or of a mixture of aFormula 2 detergent and a quaternary or tertiary ammonium cation sulfateor sulfonate salt, in combination with salt already in the buffer, mayresult in a concentration of salt as to cause the glycoprotein toprecipitate. Where the practitioner intends to perform an assay using anew combination of a particular ammonium cation detergent and aparticular buffer with a particular glycoprotein, or of a mixture of aFormula 2 detergent and an quaternary or tertiary ammonium cationsulfate or sulfonate detergent, it is good practice to combine a smallamount of these components to verify that the glycoprotein remains insolution. if the glycoprotein precipitates, which is easily observedvisually, that indicates that the particular combination is too saltyfor use with that glycoprotein and that the practitioner should select abuffer with a lower salt concentration or a different ammonium cationdetergent. As persons of skill are aware, these kinds of preliminarytests to find combinations of reagents suitable for use with aparticular glycoprotein are usual in this art.

Persons of skill will further appreciate that the ease of denaturingglycoproteins depends on a range of factors, including their secondaryand tertiary structure, and some glycoproteins are resistant todenaturing even under conditions that would denature most otherglycoproteins. In some embodiments, particularly with regard todenaturing a glycoprotein known to be hard to denature or one proving inpractice to be hard to denature completely using an ammonium cationsulfate or sulfonate detergent, the practitioner may wish to also addone or more additional detergents or additional organic solventdenaturants, such as acetonitrile or tetrahydrofuran, or a chaotrope,such as urea or guanidinium chloride.

As noted in the Background, current protocols often combine the anionicdetergent SDS with a non-ionic detergent. We have found that a nonionicdetergent is not needed with the exemplar quaternary ammoniumdetergents. Accordingly, in preferred embodiments, a non-ionic detergentis not present during deglycosylation of target glycoproteins orglycopeptides.

A detergent of Formula 2 may be mixed with more than one ammonium cationsalt at a time in an effort to improve solubilization of a glycoproteinor glycoprotein mixture for deglycosylation. Similarly, more than oneFormula 2 detergent may be used with an ammonium cation salt or two suchsalts. In some embodiments, two or three different ammonium cationdetergents may be combined in solution with the glycoprotein or mixtureof glycoproteins to be denatured. In some of these embodiments, theammonium cation of the two or the three salts is the same, but the anionis different. In other embodiments, the anion of two detergents is thesame, but one of the detergents has a tertiary ammonium cation and theother a quaternary ammonium cation, or one has one tertiary cation andthe other a different tertiary cation, or one has a first quaternarycation and the second has a second quaternary ammonium cation. Inpractice, there are diminishing returns as the number of detergentsincreases and while it might not be unusual to use two or three, it isunusual to see protocols calling for five or more.

The use of multiple detergents and salts can complicate cleanup andremoval for downstream analytic steps. In some embodiments, the ammoniumcation detergents are all quaternary ammonium sulfate detergents. Insome embodiments, the ammonium cation detergents are all quaternarysulfonate detergents. In some embodiments, the detergents are of thesame or of different types, but can be conveniently be removed by use ofa suitable cleanup column. Persons of skill are knowledgeable about theuse of these reagents and their removal after they have served theirpurpose.

Other Agents that may be used During Denaturation of the Glycoprotein

The solution containing the glycoprotein and the ammonium cationdetergent can contain further agents commonly used in protocols fordeglycosylating glycoproteins. In particular, the solution can containreductants, such as tris(2-carboxyethyl)phosphine, dithiothreitol (DTT),beta-mercaptoethanol (BME), alkylants, such as iodoacetamide, or acombination of reductants and alkylants. The solution can contain anorganic solvent denaturant, such as acetonitrile, tetrahydrofuran,trifluoroethanol, or hexafluoroisopropanol and may contain a chaotrope,such as urea or guanidinium chloride. It is expected that persons ofskill in denaturing and deglycosylating glycoproteins are familiar withthe use of each of these types of reagents and the compounds usuallyused for these purposes.

Heating the Glycoprotein-Detergent Mixture to Speed Denaturation

To denature the glycoprotein, a solution containing the ammonium cationdetergent of choice is added to the glycoprotein and the resultingmixture is incubated. In some embodiments in which the practitioner wantto complete the denaturation more quickly, the mixture can be heated. Insome embodiments, the mixture is heated to a high temperature(typically, 90° C., although for glycoproteins known to be hard todenature, it may be higher). In some embodiments, the mixture is heatedas high as 100° C. In most embodiments, the mixture will not be heatedhigher than 100° C., although in some embodiments, the mixture may beheated as high as 120′ C. Typically, the solution will be heated for atime between 1 minute and about 10 minutes, more preferably 2-7 minutes,still more preferably about 2-5 minutes, even more preferably about 3 toabout 5 minutes and most preferably about 3 minutes, It is not expectedthat heating the mixture for more than about 5 minutes will improve thedenaturation of the glycoprotein. As used herein in connection with astatement of a time, the term “about” means plus or minus 30 seconds.

Cooling

The denatured glycoprotein or glycopeptide will preferably be cooledbefore being deglycosylated by a deglycosylation enzyme, such as theexemplar deglycosylation enzyme PNGase F, both to avoid denaturing thedeglycosylation enzyme once it is added and to permit thedeglycosylation to occur at a temperature in a range at which the enzymeis most active. The solution containing the glycoprotein is preferablycooled to about 22-60° C., and more preferably about 35-55° C. In somepreferred embodiments, the glycoprotein or glycopeptide is cooled toabout 45-50° C. and most preferably about 50° C. Persons of skill willappreciate that for some equipment, the heat transfer from the apparatusto the reactants is not complete and that a temperature setting of theheating apparatus will result in the reactants being at a temperatureseveral degrees cooler than the temperature setting, and will adjustaccordingly. As used herein in connection with a temperature, the term“about” means plus or minus 1 degree C.

Deglycosylation

In some embodiments, the glycoprotein is deglycosylated by adeglycosylation enzyme. In some embodiments, the deglycosylation enzymeis an amidase. In some embodiments, the deglycosylation enzyme is theamidase PNGase F. In some embodiments, the deglycosylation enzyme is anendoglycosidase such as Endoglycosidase F1, Endoglycosidase F2,Endoglycosidase F3, or Endoglycosidase H. In some embodiments, thepractitioner wishes to distinguish between any N-glycans that may bepresent on the glycoprotein from any O-glycans that may be present. Insome embodiments, the enzymes mentioned above are used in connectionwith an ammonium cation detergent denaturation to provide a fast methodof removing the N-glycans from the glycoprotein so that any O-glycans orglycosaminoglycans (GAGS) that may be on the glycoprotein can beanalyzed. For example, the first digestion may be made using an enzymeto remove N-glycans, followed by a second enzymatic digestion withendo-alpha-N-acetyl-galactosaminidase to remove O-glycans. It isexpected that persons of skill are familiar with the various enzymesused for enzymatic release of carbohydrates from glycoconjugates ingeneral and from glycoproteins in particular.

Labeling

PNGase F, a widely used deglycosylation enzyme, releases glycans fromglycoproteins as glycosylamines. Various methods of labelingglycosylamines are known in the art, as exemplified by co-owned U.S.Pat. Nos. 8,124,792 and 8,445,292. If the glycosylamines are to belabeled with an amine-reactive dye, the dye labeling can be conductedwithout removal of the ammonium cation detergent. If labeling is to beperformed using reductive amination of the reducing end of a glycan,rather than by releasing them from a glycoprotein as glycosylamines, theammonium cation detergent is preferably first removed. Regardless of themethod of labeling, the ammonium cation detergent may be removed beforesubjecting the labeled glycans or unlabeled glycoprotein to analyticalmethods, such as mass spectrometry, in which the presence of theammonium cation detergent might be incompatible or would be aconfounding factor.

Removal of the Detergents

Detergents used in deglycosylation protocols are preferably removedafter labeling but before analysis of the labeled glycans, of thelabeled glycosylamines, or of the glycoprotein or glycopeptide (which,following the deglycosylation step, is deglycosylated or aglycosylated),as detergents can be incompatible with some analytical instruments.Quaternary ammonium fatty acid detergents can be removed byprecipitation with an acid, leaving behind in solution thenow-deglycosylated or aglycosylated protein and the glycans releasedfrom the glycoprotein behind in the supernatant. The supernatant canthen be removed by, for example, pipetting the supernatant away from theprecipitate.

Detergents of Formula 1 or Formula 2 can be removed by a solid phaseextraction called hydrophilic interaction liquid chromatography, or“HILIC.” In studies conducted with an exemplar quaternary ammoniumsulfate detergent, we found that HILIC was very effective at removingdetergent from the deglycosylation solution. HILIC is a preferredembodiment for removal of ammonium sulfate or ammonium sulfonatedetergents of Formula 1 used in some embodiments of the invention.

In some embodiments, the detergent or detergents may be removed by usingother solid- or liquid-phase techniques. It is expected that persons ofskill are familiar with various types of liquid-liquid techniques andsolid phase extraction devices which are used in the art to removedetergents from a solution. The solid phase extraction devices usuallycomprise resins on a solid support, and the resins are convenientlydisposed in a cartridge or column (for convenience of reference,reference to a “column” in the following discussion refers to either acolumn or a cartridge, unless otherwise required by context). Commonsolid phase extraction devices include reverse phase columns, normalphase cartridges or columns, ion exchange columns, and size exclusioncolumns. Typically, the cleanup columns bind the glycans, allowing thedetergent to flow through and be discarded, after which the glycans areeluted from the column. The use of a solid- or liquid-phase extractiontechnique is particularly preferred when the ammonium cation detergentis not susceptible to acid precipitation or to ensure removal of anydetergent that does not precipitate out of solution.

If one or more denaturants are used in addition to an ammonium cationdetergent, they are typically also removed by using a cleanup column,such as a solid phase extraction column, and do not have to be LC or MScompatible.

Analysis of Glycans, Glycoproteins, or Both

Glycans can typically be eluted from a solid phase extraction devicewith water, after which they can be put in an analytical column orsubjected to mass spectrometry (“MS”). Typical analytical means foranalyzing labeled glycans or glycosylamines include high-pressure liquidchromatography, capillary electrophoresis, fluorescence analysis, massspectrometry, or a combination of two or more of any of these. In someembodiments, the combination is of fluorescence analysis and massspectrometry. Glycoproteins or glycopeptides deglycosylated during thecourse of releasing the glycans can themselves be analyzed, by anyconvenient means, for example, high-performance liquid chromatography,hydrophilic interaction chromatography, nuclear magnetic resonance,Western blotting, gel electrophoresis, fluorescence analysis, capillaryelectrophoresis, microfluidic separation, mass spectrometry, or acombination of two or more of any of these.

EXAMPLES Example 1

This Example sets forth the method used to prepare an exemplarquaternary ammonium sulfate detergent, tetramethyl ammonium dodecylsulfate.

Twenty mL of AMBERLYST® 36 (Sigma-Aldrich, St. Louis, Mo.), acation-exchange resin, was loaded into a glass chromatography columnwith a coarse frit and rinsed with ten column volumes of water.Tetramethylammonium hydroxide was passed through the column as anaqueous solution, and the eluent was monitored with pH paper todetermine the transition from neutral to basic pH, Excesstetramethylammonium hydroxide was rinsed from the column with water, andan aqueous solution of sodium dodecyl sulfate was loaded onto thecolumn. The molar amount of SDS loaded was approximately tenfold lessthan the molar amount of tetramethylammonium ions estimated to bepresent on the resin. The column was flushed with water. Finally, thewater was removed by rotary evaporation, yielding a white solid composedof tetramethylammonium dodecyl sulfate.

Example 2

This Example sets forth the protocol used for comparisons of the amountof glycan released from glycoproteins by enzymatic digestion followingdenaturing with either (a) sodium dodecyl sulfate or (b) an exemplarquaternary ammonium sulfate detergent, tetramethyl ammonium dodecylsulfate.

Denaturation Step

A series of glycoproteins were selected for testing ranging from havingone glycosylation to multiple sites. Aliquots of 40 to 100 μg solutionof each glycoprotein to be tested were prepared in 20 μl of pH 7.5 in acompatible reaction buffer of choice, forming a solution containingglycoprotein and water. Two μl of a 50 mM solution of either sodiumdodecyl sulfate (detergent 1) or tetramethyl ammonium dodecyl sulfate(detergent 2) were added to the glycoprotein/buffer solution for a finalconcentration of 4.5 mM of the detergent. The mixtures were thenincubated for 3 minutes at 90° C.

Enzymatic Digestion Step

Two μl of 1 mg/ml of PNGase F in tetramethyl ammonium HEPES buffer, pH8,was added and the resulting mixture was incubated for at 37° C. for onehour.

Labeling Step

Following this incubation, glycans released by the PNGase F were labeledwith InstantPC® dye (ProZyme, Inc., Hayward, Calif.) by adding 5 μl ofthe dye solution to each sample and incubating the samples for oneminute at 50° C.

Cleanup Step

The labeled glycans were resuspended in 600 μl of 95:5 acetonitrile:formic acid and the solutions were loaded onto cleanup cartridges. Thecartridges were then washed three times with 600 μl of a 95:5acetonitrile: formic acid solution. The labeled glycans were then elutedwith 100 μl of a solution of 200 mM ammonium formate, pH 7, containing10% acetonitrile.

Analysis Step

One μl of each eluted sample was injected into high performance liquidchromatography (HPLC) equipment for analysis. The amount of glycansreleased from each glycoprotein when tetramethyl ammonium dodecylsulfate was used as the detergent was considered to be “100%” release,which allowed a ready comparison to the amount of glycan released usingthe same assay on the same glycoprotein, but using sodium dodecylsulfate rather than tetramethyl ammonium dodecyl sulfate as thedetergent to denature the glycoprotein.

Example 3

This Example sets forth the results of the study reported in Example 2.

FIG. 1 is a table setting forth the results of studies comparing the useof tetramethyl ammonium dodecyl sulfate and sodium dodecyl sulfate (SDS)as detergents in a deglycosylation protocol on a series of nineglycoproteins and mixtures of glycoproteins. To permit ready comparisonof how well each detergent served in the deglycosylation protocol, theamount of glycans released from each glycoprotein or mixture ofglycoproteins in the assay in which tetramethyl ammonium dodecyl sulfatewas used as the detergent was normalized to 100%, and the amountreleased then compared to the amount of glycan released when SDS wasused as the detergent in the same deglycosylation protocol on the sameglycoprotein. The studies included three mixtures of human antibodies ofdifferent antibody classes: hIgG, hIgA, and hIgM; an antibody,designated RM 8671, provided by the National Institute of Standards andTechnology (“NIST”) as a reference material for testing, and fiveglycoproteins currently approved by the Food and Drug Administration fortherapeutic use. FIG. 2 presents the results of the comparative studiesreported in FIG. 1 in the form of a graph, with error bars representingthe results of three replicates. To allow ready comparison, the amountof glycans released from each glycoprotein or mixture by digestionfollowing denaturing with tetramethyl ammonium lauryl sulfate wasconsidered to be a 100% release, and that amount was then compared tothe amount released from the same glycoprotein or glycoprotein mixturefollowing denaturing with SDS.

For four out of nine of the glycoproteins or glycoprotein mixtures,denaturing with SDS resulted in the release of significantly fewerglycans than did the use of the exemplar quarternary ammonium sulfatedetergent, and for the mixture of human IgGs, denaturing with SDSresulted in the release of almost 25% less glycan than did denaturingwith the quarternary ammonium sulfate detergent. For a mixture of hIgMantibodies, denaturing with SDS resulted in the release of 18% lessglycan than did denaturing with the quarternary ammonium sulfatedetergent. For the therapeutic biologic agent ORENCIA®, denaturing withSDS resulted in the release of almost 15% less glycan than diddenaturing with the quarternary ammonium sulfate detergent. For three ofthe nine glycoproteins or mixtures, using SDS to denature theglycoprotein resulted in roughly the same release of glycan comparedwith the amount released following denaturation of the same glycoproteinwith the quarternary ammonium sulfate detergent. In the case of oneglycoprotein, the chimeric mouse/human monoclonal antibody cetuximab,denaturing with SDS resulted in the release of more glycan than did theuse of quarternary ammonium sulfate detergent, and in that case thedifference was less than 10%. The results indicate that quarternaryammonium sulfate detergents provide generally better or equal results,and in many cases, surprisingly better results in deglycosylationprotocols compared to SDS.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-50. (canceled)
 51. A solution for denaturing a glycoprotein orglycopeptide of interest in vitro, said solution comprising (A) saidglycoprotein or glycopeptide of interest and (B) an effective amount ofan ammonium cation sulfate or sulfonate detergent comprising (a) analiphatic chain of 8-24 carbons, (b) a sulfate or sulfonate anion and(c) a tertiary or quaternary ammonium cation, wherein said aliphaticchain is covalently attached to said anion.
 52. The solution of claim51, wherein said aliphatic chain of 8-24 carbons is saturated.
 53. Thesolution of claim 51, wherein said aliphatic chain is 12 carbons. 54.The solution of claim 51, wherein said cation is a quaternary ammoniumcation.
 55. The solution of claim 54, wherein said quaternary ammoniumcation is selected from the group consisting of:


56. The solution of claim 55, wherein said quaternary ammonium cation isa tetramethylammonium cation.
 57. The solution of claim 51, wherein saidammonium cation sulfate detergent is tetramethyl ammonium dodecylsulfate.
 58. The solution of claim 51, wherein said cation is a tertiaryammonium cation.
 59. The solution of claim 51, further comprising adeglycosylation enzyme.
 60. The solution of claim 51, further whereinsaid solution comprises a reductant, an alkylant, an additional organicsolvent denaturant, an additional detergent, a chaotrope, or acombination of any of these.
 61. A kit for denaturing anddeglycosylating a glycoprotein or glycopeptide of interest in vitro,said solution comprising (A) an ammonium cation sulfate or sulfonatedetergent comprising (a) an aliphatic chain of 8-24 carbons, (b) asulfate or sulfonate anion and (c) a tertiary or quaternary ammoniumcation, wherein said aliphatic chain is covalently attached to saidanion, and (B) a reductant, an alkylant, an additional organic solventdenaturant, an additional detergent, a chaotrope, or a combination ofany of these.
 62. The kit of claim 61, further comprising adeglycosylation enzyme.
 63. The kit of claim 62, further wherein saiddeglycosylation enzyme is PNGase F.
 64. The kit of claim 61, whereinsaid aliphatic chain is of 12 carbons.
 65. The kit of claim 61, whereinsaid cation is a quaternary ammonium cation.
 66. The kit of claim 65,wherein said quaternary ammonium cation is selected from the groupconsisting of:


67. The kit of claim 66, wherein said quaternary ammonium cation is atetramethylammonium cation.
 68. The kit of claim 61, wherein saidammonium cation sulfate detergent is tetramethyl ammonium dodecylsulfate.
 69. The kit of claim 61, wherein said cation is a tertiaryammonium cation.
 70. The kit of claim 69, wherein said tertiary ammoniumcation is a trimethyl, tributyl, triethyl, or tripropyl ammonium cation.